The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV–growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.
The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana.We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.
In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.
Phytochromes (phy) are red/far-red-absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N-and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.Arabidopsis | basic helix-loop-helix | signal transduction P lant development is strictly regulated by the environmental light conditions. Even though light is ubiquitously present, spectral composition, intensity, and light direction varies depending on the local environment. To adapt on local light conditions plants are equipped with a highly developed repertory of photoreceptors (1, 2).Plant phytochromes are mainly involved in detection of red and far-red light wavelengths of the spectrum. A family of five members in Arabidopsis thaliana regulates the major developmental steps in the life of the plant: germination, photomorphogenesis, and flowering (3, 4). Despite the sequence similarities and identical spectroscopic features, the two major phytochromes, phyA and phyB, show overlapping as well as unique photosensory and functional characteristics (5-7). The light-stable phyB mediates red/far-red reversible low fluence response, whereas the light-labile phyA, is involved in far-red light sensing (high irradiance response) and in responses to very weak light (very low fluence response, VLFR) (8).Phytochromes are cytosolic proteins of 125 kDa that are translocated into the nucleus upon light activation (6, 9-11). Nuclear transport is an essential step in phytochrome signaling and a prerequisite for the interaction with transcription factors in the nucleus (12, 13). Concerning the kinetics of nuclear import, severe differences can be found between the two major plant phytochromes, phyA and phyB (6). PhyA cannot be detected in nuclei of dark-grown plants but is rapidly transported into the nucleus upon light irradiation. In contrast, low levels of phyB are present in the nucleus even in dark-grown seedlings. The nuclea...
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